The objective of this proposal is to understand ameloblast cell differentiation during amelogenesis. We propose to use specific markers, unique for differentiated ameloblasts, for these studies. We have chosen the enamelin proteins as specific markers, and the rabbit tooth organ as our experimental system, because it is highly enriched for this class of proteins (36-40% of total proteins extracted). First, we will characterize the enamelin proteins by their physico-chemical properties. Enzymatical and chemical cleavage of the original protein into smaller peptides will be used for determination of their primary structure. Polyclonal antibodies specific for enamelins will be produced. Other available antibodies for enamelins cross-react with amelogenins. Therefore, peptides unique for enamelin proteins will be used to immunize guinea pigs and obtain enamelin-specific antibodies. The antibodies will be used to detect the presence of enamelin polypeptides during tooth morphogenesis; to characterize the enamelin mRNAs; and to characterize bacterial clones containing enamelin cDNA sequences. In additional studies, we propose to isolate and characterize the mRNAs responsible for the synthesis of enamelin proteins. These mRNAs will be identified by their translational activity in a cell-free system, and the products translated will be characterized by immunoprecipitation with enamelin antibodies. The total poly(A)-containing RNAs will be used to produce cDNAs with the enzyme reverse transcriptase. This enzyme will also produce a double stranded-cDNA which will be inserted into a plasmid and amplified in a bacterial host. The clones containing enamelin-cDNA sequences will be identified by colony hybridization, hybrid-arrested and hybrid-selected translation. These clones will be the source of specific probes to detect enamelin gene transcription during tooth organogenesis by in situ hybridization techniques. These probes will also be used to analyze the structure and organization of the enamelin genes. Nucleotide sequence analysis, Southerns and R-looping techniques will be used for these studies.